The invention relates to novel calpains and to their preparation. The invention furthermore relates to methods for screening for novel calpain inhibitors and to their use.
Calpains belong to the intracellular, non-lysosomal enzymes of the cysteine protease group. They are involved in Ca2+-dependent signal transduction in eukaryotic cells, ie. they control cellular functions depending on the Ca2+ concentration. Calpains occur ubiquitously in animal tissues and cells from, for example, humans, chickens, rabbits or rats. Calpains have also been found in lower animals, for example in Drosophila melanogaster or Caenorhabditis elegans. No calpains have yet been detected in yeasts, fungi or bacteria.
To date, three main isoforms of these ubiquitous calpains have become known and are distinguished in vitro by their calcium-dependent activability. Calpain I (=xcexccalpain) is activated by xcexc-molar calcium ion concentrations, while calpain II (=mcalpain) is activated only by millimolar concentrations of calcium ions. Both calpains consist of two subunits, one large subunit of about 80 kDa and one small subunit of about 30 kDa. Both subunits of the active heterodimer have binding sites for calcium. The large subunit is composed of the following four protein domains (=I-IV): a protease domain (=domain II), a calcium-binding domain (=domain IV) and two other domains (=domain I and III) whose function is unclear. The small 30 K 30 subunit consists of a calcium-binding subunit (=IVxe2x80x2) and of another subunit (=V) whose function is unclear. In addition to these two calpain types, a third type, which is intermediate with respect to calcium activation (=xcexc/m 80K), has been found in chickens (Wang K. K. W. et al., TiPS, Vol. 15, 1994: 412-419, Suzuki, K et al., Biol. Chem. Hoppe-Seyler, Vol. 376, 1995: 523-529).
Besides these ubiquitously occurring calpains, recently two new calpains whose expression is tissue-specific have been identified. nCL-1 (=p94) is a muscle-specific calpain which occurs in chickens, rats and humans and which might be active as monomer and consists only of the 80 kd subunit. Besides nCL-1 there is a stomach-specific calpain which may occur in two splicing variants nCL-2 and nCL-2xe2x80x2. nCL-2xe2x80x2 differs from nCL-2 by the absence of the calcium-binding region (Sorimachi, H. S. et al., J. Biol. Chem. Vol. 268, No. 26, 1993: 19476-19482, Sorimachi, H. S. et al., FEBS Lett. 343, 1994: 1-5). A calpain-homologous protein (=CalpA) which interacts with actin and presumably plays an important part in embryonic development, and which displays two different splicing variants, has also been found in Dorosophila (Mol. Cell. Biol. Vol. 15, No. 2, 1995: 824-834). In this case too, the shorter variant lacks the calcium-binding site.
It is presumed that calpains play important parts in various physiological processes. A large number of cytoskeletal, membrane-bound or regulatory proteins such as protein-kinase C, phospholipase C, spectrin, cytoskeletal proteins such as MAP2, muscle proteins, neurofilaments and neuropeptides, platelet proteins, epidermal growth factor, NMDA receptor and proteins involved in mitosis, and other proteins, are calpain substrates (Barrett M. J. et al., Life Sci. 48, 1991: 1659-69, Wang K. K. et al., Trends in Pharmacol. Sci., 15, 1994: 412-419). The normal physiological function of the calpains is stil not even now clearly understood.
Elevated calpain levels have been measured in various pathophysiological processes and diseases, for example in: ischemias of the heart (eg. myocardial infarct), of the kidney or of the central nervous system (eg. stroke), inflammations, muscular dystrophies, cataracts of the eyes (gray cataract), injuries to the central nervous system (eg. trauma), Alzheimer""s disease, HIV-induced neuropathy, Parkinson""s and Huntigton""s [sic] diseases etc. (see Wang K. K. above). It is presumed that these diseases are connected with an elevated and persistent intracellular calcium level. This results in overactivation of calcium-dependent processes which are then no longer subject to physiological regulation. Accordingly, overactivation of calpains may also induce pathophysiological processes.
It has therefore been postulated that inhibitors of calpain enzymes may be useful for treating these diseases. Various investigations have confirmed this. Thus, Seung-Chyul Hong et al. (Stroke 1994, 25 (3), 663-669) and Bartus R. T. et al. (Neurological Res. 1995, 17, 249-258) show that calpain inhibitors have a neuroprotective effect in acute neurodegenerative disorders occurring after stroke. Likewise, calpain inhibitors improve the recovery from the memory deficits and neuromotor disturbances occurring after experimental brain traumas (Saatman K. E. et al., Proc. Natl. Acad. Sci. USA, 93, 1996: 3428-3433). Edelstein C. L. et al. (Proc. Natl. Acad. Sci. USA, 92, 1995, 7662-7666) found a protective effect of calpain inhibitors on hypoxia-damaged kidneys. Yoshida K. I. et al. (Jap. Circ. J. 59 (1), 1995, 40-48) were able to show beneficial effects of calpain inhibitors after cardiac damage caused by ischemia or reperfusion. Since calpain inhibitors inhibit the release of xcex2-AP4 protein, a potential use for treating Alzheimer""s disease has been proposed (Higaki J. et al., Neuron, 14, 1995: 651-659). The release of interleukin-1xcex1 is likewise inhibited by calpain inhibitors (Watanabe N. et al., Cytokine, 6 (6), 1994: 597-601). It has furthermore been found that calpain inhibitors show cytotoxic effects on tumor cells (Shiba E. et al., 20th Meeting Int. Ass. Breast Cancer Res., Sendai Jp, 1994, 25th-28Sept., Int. J. Onco. 5 (Suppl.), 1994, 381). Calpain also plays an important part in restenosis and in arthritis, and calpain inhibitors may have a beneficial effect on the pathology (March K. L. et al. Circ. Res. 72, 1993: 413-423, Suzuki K. et al., Biochem J., 285, 1992: 857-862).
Further possible uses of calpain inhibitors are to be found in Wang K. K. (Trends in Pharmacol. Sci., 15, 1994: 412-419).
The most potent and selective calpain inhibitor is the naturally occurring intracellular protein calpastatin. It inhibits both calpain I and calpain II, but not other cysteine proteases or thiol proteases such as cathepsin B, L or papain. However, the disadvantage of calpastatin, which consists of about 700 amino acids, is that because of the size and the inability to pass through the cell membrane it is unsuitable for possible therapies. Besides calpain inhibitors which are low molecular weight peptides, a number of non-peptide inhibitors has been identified. The disadvantages of these inhibitors are that they are unstable, are rapidly metabolized and, in some cases, are toxic. Many calpain inhibitors additionally display inusfficient selectivity, ie. they inhibit not only calpain I and II but also other cysteine proteases such as papain, chymotrypsin, elastase or cathepsin B and L.
Thus there still remains a need for selective, highly effective calpain inhibitors. Highly specific test systems which allow selective inhibitors to be identified are needed to screen for these selective, highly effective calpain inhibitors. These screening tests are normally carried out with the ubiquitously occurring calpains I and II.
To find selective inhibitors, it is necessary and desirable to provide for testing further calpains which are, if possible, expressed tissue-specifically so that the inhibitors can be tested for their selectivity between the individual calpains.
In addition, further new calpains are sought-after proteins because it is highly probable that they are expressed differently in different pathologies or diseases and play an important part in these diseases.
It is an object of the present invention to provide means for profiling and identifying calpain inhibitors which make it possible to identify calpain inhibitors which, on the one hand, have an inhibitory effect on only one calpain and, on the other hand, have an inhibitory effect on several calpains, and to provide these as a therapeutic target.
We have found that this object is achieved by a novel calpain and its allelic variants, analogs or derivatives.
The invention also relates to a method for identifying calpain inhibitors, wherein the calpain nCL-3 encoded by the sequence SEQ ID NO:1 or SEQ ID NO:6 is isolated from tissues or cells in which the enzyme nCL-3 is expressed, and the inhibition of the cleavage of a substrate of the enzyme nCL-3 and, in at least one other test, the inhibition of the cleavage of a substrate of the enzymes calpain I and/or II by test substances are measured, and the test substances which show an inhibitory effect on at least one of the calpains are selected, or the test substances which do not inhibit the enzyme nCL-3 but the enzymes calpain I and/or II or which inhibit the enzyme nCL-3 but not the enzymes calpain I and/or II or which inhibit nCL-3 and the enzymes calpain I and/or II are selected.
The invention furthermore relates to a method for identifying calpain inhibitors which comprises determining the inhibition of the cleavage of a substrate of the enzyme nCL-3 or of calpains I and/or II by test substances in cellular systems, and selecting those test substances which pass through the cell membrane and which inhibit the intracellular activity of the enzyme nCL-3 and/or of the calpains I and/or II.